Fluorescent dyes are widely used as indispensable markers in biology, optical microscopy, and analytical chemistry.
In particular, the sensitive and stable imaging of cellular components depends on the favorable combination of chemical, biological and physical factors. The availability and proper choice of fluorescent dyes is a key factor to success of the entire labeling and imaging procedure. Due to their superior brightness and photostability, synthetic dyes represent an attractive alternative to fluorescent proteins.
Notwithstanding the large number of fluorescent dyes which are known and used for various labeling and imaging applications there is still a need for the new dyes possessing compact structures, increased Stokes shifts (separation between the absorption and emission maxima) and reactive groups. Dyes with compact structures and a zero net charge (neutral molecules or zwitterionic species with a short charge separation distance) are known to penetrate the outer plasma membrane of living cells and may be used as fluorescent labels in biology, optical microscopy and life sciences, when the imaging of living specimen is required. For example, among the multitude of fluorescent dyes reported hitherto, only triarylmethane dyes of rhodamine (see a) A. N. Butkevich, G. Y. Mitronova, S. C. Sidenstein, J. L. Klocke, D. Kamin, D. N. Meineke, E. D'Este, P. T. Kraemer, J. G. Danzl, V. N. Belov, S. W. Hell, Angew. Chem. Int. Ed. 2016, 55, 3290-3294; b) F. Bottanelli, E. B. Kromann, E. S. Allgeyer, R. S. Erdmann, S. Wood Baguley, G. Sirinakis, A. Schepartz, D. Baddeley, D. K. Toomre, J. E. Rothman, J. Bewersdorf, Nat. Commun. 2016, 7, 10778; c) B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, Biophys. J. 2010, 98, 158-163; d) S. C. Sidenstein, E. D'Este, M. J. Bohm, J. G. Danzl, V. N. Belov, S. W. Hell, Sci. Rep. 2016, 6, 26725), carborhodamine (A. N. Butkevich, G. Y. Mitronova, S. C. Sidenstein, J. L. Klocke, D. Kamin, D. N. Meineke, E. D'Este, P. T. Kraemer, J. G. Danzl, V. N. Belov, S. W. Hell, Angew. Chem. Int. Ed. 2016, 55, 3290-3294; and silicon-rhodamine (SiR) (see a) G. Lukinavicius, K. Umezawa, N. Olivier, A. Honigmann, G. Yang, T. Plass, V. Mueller, L. Reymond, I. R. Correa, Jr., Z. G. Luo, C. Schultz, E. A. Lemke, P. Heppenstall, C. Eggeling, S. Manley, K. Johnsson, Nat. Chem. 2013, 5, 132-139; b) G. Lukinavicius, L. Reymond, E. D'Este, A. Masharina, F. Gottfert, H. Ta, A. Guther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H. D. Arndt, S. W. Hell, K. Johnsson, Nat. Methods 2014, 11, 731-733; c) G. Lukinavicius, L. Reymond, K. Umezawa, O. Sallin, E. D'Este, F. Gottfert, H. Ta, S. W. Hell, Y. Urano, K. Johnsson, J. Am. Chem. Soc. 2016, 138, 9365-9368; d) Y. Kushida, T. Nagano, K. Hanaoka, Analyst 2015, 140, 685-695) classes bearing a carboxyl in the ortho-position of the pendant aromatic ring have been shown to provide specific vital labeling and perform well in superresolution fluorescence microscopy. Importantly, all these dyes feature small Stokes shifts of 20-40 nm. However, fluorescent dyes with increased Stokes shifts offer an advantage of using more flexible imaging schemes (when combined with small Stokes shift dyes). In this case two fluorescent labels (with small and large Stokes shifts) can be combined in one experiment and imaged separately; either by using two excitation sources and one detection channel, or by applying one excitation wavelength and two detection windows. Therefore, the discovery of new low molecular weight (MW) labels with increased Stokes shifts and sufficient emission efficiencies (because the very large Stokes shift (>100 nm) dyes typically demonstrate low fluorescence quantum yields, especially in aqueous media) is a vital task in modern biology-related natural science.
Another drawback persistent in bioconjugation techniques is that many widely used and bright lipophilic triarylmethanes are cationic dyes and bind non-specifically and stain membrane structures (L. C. Zanetti-Domingues, C. J. Tynan, D. J. Rolfe, D. T. Clarke, M. Martin-Fernandez, PLoS One 2013, 8, e74200).
On the other hand, numerous anionic fluorescent dyes, commercially available as sulfonates or phosphates, are hydrophilic and highly water-soluble but do not penetrate the intact plasma membrane. These labels are used nearly exclusively in immunostaining of fixed cells, and delivery of cell-impermeant labels into living cells requires the use of sophisticated techniques such as conjugation with membrane-permeant peptides (see a) Z. Qian, A. Martyna, R. L. Hard, J. Wang, G. Appiah-Kubi, C. Coss, M. A. Phelps, J. S. Rossman, D. Pei, Biochemistry 2016, 55, 2601-2612; b) J. P. Richard, K. Melikov, E. Vives, C. Ramos, B. Verbeure, M. J. Gait, L. V. Chernomordik, B. Lebleu, J. Biol. Chem. 2003, 278, 585-590; c) M. Silhol, M. Tyagi, M. Giacca, B. Lebleu, E. Vives, Eur. J. Biochem. 2002, 269, 494-501) or reversible membrane permeabilization (K. W. Teng, Y. Ishitsuka, P. Ren, Y. Youn, X. Deng, P. Ge, A. S. Belmont, P. R. Selvin, eLife 2016, 5, e20378).
In view of the drawbacks of fluorescent dyes of the prior art and the alternative strategies to obviate the limitations of their delivery to living cells and tissues, the main object of the present invention is to provide a novel general approach to dyes, in particular fluorescent dyes with superior properties such as:                1. neutral (with a zero net charge) or zwitterionic with a very short charge separation distance;        2. limited molecular mass (preferably with MW <500-700) and a compact structure;        3. bathochromic and bathofluoric shifts of absorption and emission bands, with the emission of light preferably in the red spectral region (>600 nm);        4. easy introduction of additional functional groups (e.g., carboxylate groups for further conjugation);        5. increased Stokes shifts (with sufficient emission efficiency).        
This objective has been achieved by providing novel compounds and fluorescent dyes according to claims 1-9, a method for preparing the same according to claim 11, conjugates comprising said compounds and dyes according to claim 11, as well as the applications of the disclosed novel compounds according to claims 15 to 18. The newly introduced labels satisfy all of the above-stated requirements. Further aspects and more specific embodiments of the invention are the subject of further claims.